Direct visualization of epithelial microvilli biogenesis.

Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also collapse, characterized first by loss of membrane wrapping and ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS levels, and ultimately disassembly of the core actin bundle itself. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts.

PACSIN2-dependent apical endocytosis regulates the morphology of epithelial microvilli.

Apical microvilli are critical for the homeostasis of transporting epithelia, yet mechanisms that control the assembly and morphology of these protrusions remain poorly understood. Previous studies in intestinal epithelial cell lines suggested a role for the F-BAR domain protein PACSIN2 in normal microvillar assembly. Here we report the phenotype of PACSIN2 KO mice and provide evidence that through its role in promoting apical endocytosis, this molecule plays a role in controlling microvillar morphology. PACSIN2 KO enterocytes exhibit reduced numbers of microvilli and defects in the microvillar ultrastructure, with membranes lifting away from rootlets of core bundles. Dynamin2, a PACSIN2 binding partner, and other endocytic factors were also lost from their normal localization near microvillar rootlets. To determine whether loss of endocytic machinery could explain defects in microvillar morphology, we examined the impact of PACSIN2 KD and endocytosis inhibition on live intestinal epithelial cells. These assays revealed that when endocytic vesicle scission fails, tubules are pulled into the cytoplasm and this, in turn, leads to a membrane-lifting phenomenon reminiscent of that observed at PACSIN2 KO brush borders. These findings lead to a new model where inward forces generated by endocytic machinery on the plasma membrane control the membrane wrapping of cell surface protrusions.

Loss of myosin Vb promotes apical bulk endocytosis in neonatal enterocytes.

In patients with inactivating mutations in myosin Vb (Myo5B), enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis, the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus, apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes.

Actin Dynamics Drive Microvillar Motility and Clustering during Brush Border Assembly.

Transporting epithelial cells generate arrays of microvilli, known as a brush border, to enhance functional capacity. To understand brush border formation, we used live cell imaging to visualize apical remodeling early in this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at âˆ¼0.2 µm/min. Perturbation with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed ends of core bundles, which in turn is linked to robust treadmilling of these structures. Actin regulatory factors IRTKS and EPS8 localize to the barbed ends of motile microvilli, where they control the kinetics and nature of movement. As the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately clustering and consolidation into higher-order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.

IRTKS (BAIAP2L1) Elongates Epithelial Microvilli Using EPS8-Dependent and Independent Mechanisms.

Transporting epithelial cells like those that line the gut build large arrays of actin-supported protrusions called microvilli, which extend from the apical surface into luminal spaces to increase functional surface area. Although critical for maintaining physiological homeostasis, mechanisms controlling the formation of microvilli remain poorly understood. Here, we report that the inverse-bin-amphiphysin-Rvs (I-BAR)-domain-containing protein insulin receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1) promotes the growth of epithelial microvilli. Super-resolution microscopy and live imaging of differentiating epithelial cells revealed that IRTKS localizes to the distal tips of actively growing microvilli via a mechanism that requires its N-terminal I-BAR domain. At microvillar tips, IRTKS promotes elongation through a mechanism involving its C-terminal actin-binding WH2 domain. IRTKS can also drive microvillar elongation using its SH3 domain to recruit the bundling protein EPS8 to microvillar tips. These results provide new insight on mechanisms that control microvillar growth during the differentiation of transporting epithelial cells and help explain why IRTKS is targeted by enteric pathogens that disrupt microvillar structure during infection of the intestinal epithelium.